Skip to main content
Download PDF

Lower genomic stability of induced pluripotent stem cells reflects increased non-homologous end joining

Cancer Communications volume 38, Article number: 49 (2018) Cite this article

Abstract

Background

Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) share many common features, including similar morphology, gene expression and in vitro differentiation profiles. However, genomic stability is much lower in iPSCs than in ESCs. In the current study, we examined whether changes in DNA damage repair in iPSCs are responsible for their greater tendency towards mutagenesis.

Methods

Mouse iPSCs, ESCs and embryonic fibroblasts were exposed to ionizing radiation (4 Gy) to introduce double-strand DNA breaks. At 4 h later, fidelity of DNA damage repair was assessed using whole-genome re-sequencing. We also analyzed genomic stability in mice derived from iPSCs versus ESCs.

Results

In comparison to ESCs and embryonic fibroblasts, iPSCs had lower DNA damage repair capacity, more somatic mutations and short indels after irradiation. iPSCs showed greater non-homologous end joining DNA repair and less homologous recombination DNA repair. Mice derived from iPSCs had lower DNA damage repair capacity than ESC-derived mice as well as C57 control mice.

Conclusions

The relatively low genomic stability of iPSCs and their high rate of tumorigenesis in vivo appear to be due, at least in part, to low fidelity of DNA damage repair.

Background

Embryonic stem cells (ESCs) are pluripotent and could differentiate into all types of somatic cells [1]. ESCc have enormous potential in the treatment of a variety of diseases, but their clinical application has been limited by ethical controversy. In 2006, Yamanaka and colleagues overexpressed four transcription factors (Oct4, Sox2, c-Myc and Klf4) in mouse somatic cells and obtained ESC-like pluripotent stem cells, termed induced pluripotent stem cells (iPSCs) [2]. iPSCs resemble ESCs in morphology, gene expression profile, epigenetic status and in vitro differentiation capacity. The development of iPSCs raises new hope for personalized clinical therapy [3,4,5].

The four transcription factors (Oct4, Sox2, c-Myc and Klf4) that are critical for the production of iPSCs are frequently overexpressed in various cancers, and mice derived from iPSCs are prone to develop tumors [6,7,8,9]. Although only a small population of transformed cells with genetic mutations is likely to develop into tumors [10], the genomic instability of iPSCs is a major concern that could produce huge impact on their eventual clinical use [11,12,13,14,15,16].

One possible explanation for the observed greater genomic instability of iPSCs is alterations in the fidelity of DNA repair pathways. Double-stranded DNA breaks, for example, can be repaired via homologous recombination (HR) with high fidelity, or via non-homologous end joining (NHEJ) with lower fidelity [17,18,19,20]. In the current study, we examined whether iPSCs differ from other types of pluripotent cells in their ability to perform these types of DNA repair. Briefly, ionizing radiation was used to induce double-stranded DNA breaks in the following cells: mouse iPSCs induced using lentivirus (lv-iPSCs) or chemically with CHR99021, Repsox and forskolin (ci-iPSCs) [21]; mouse ESCs; and mouse embryonic fibroblasts (MEFs) [22,23,24,25,26].

The experiments showed that lv-iPSCs are more likely than the other cell types to harbor genomic abnormalities, likely due to lower genomic fidelity of DNA damage repair. We also found greater genomic stability in ci-iPSCs than lv-iPSCs.

Methods

Cell lines and culture

The lv- and ci-iPSCs were derived from female transgenic OG2 mice carrying an Oct4-GFP transgene. Both types of iPSCs and ESCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 15% fetal bovine serum (FBS; Gibco), 1% MEM non-essential amino acids (Gibco), 1% penicillin/streptomycin (Gibco), 2 mmol/L l-glutamine (Gibco), 1 × 103 units/mL of mouse leukemia inhibitory factor (Millipore, Temecula, CA, USA) and 0.1 mmol/L 2-mercaptoethanol (Gibco) [27]. The medium was changed daily, and cells were passaged every 2 days using 0.25% trypsin (Thermo Fisher Scientific, Beijing, China) [28]. MEFs were cultured in DMEM supplemented with 15% FBS, 1% non-essential amino acids and 1% penicillin/streptomycin [29].

Irradiation

Cells were passaged 1 day before γ-irradiation (4 Gy) with a cobalt irradiator (Thermo Fisher Scientific). After the irradiation, cells were immediately returned to the incubator, and cultured for 4 h prior to analyses as described below.

Western blotting

To test the phosphorylation level of ATM, cells were lysed in ATM lysis buffer [20 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 0.2% Tween-20, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 2 mmol/L dithiothreitol, 50 mmol/L NaF, 500 μmol/L NaVO4, 1 mmol/L phenylmethylsulfonyl fluoride, 0.1 μg/mL aprotinin and 0.1 µg/mL leupeptin], and centrifuged, as describe previously [30].

In assays of histone modification, cells were re-suspended in 1-mL triton extraction buffer (TEB) containing 0.5% Triton X-100 and 2 mmol/L PMSF, and then lysed on ice for 10 min. The lysates were centrifuged at 1500g for 10 min at 4 °C. The pellet was washed with 1.5-mL TEB, re-suspended in 0.2 mol/L HCl, and incubated at 4 °C overnight. Samples were centrifuged at 6500g for 10 min, after which 200-µL supernatant was transferred to a new tube, and neutralized with 20-µL 2 mol/L NaOH.

Samples were separated using SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Blots were incubated with a primary antibody against one of the following proteins: phospho-ATM (1:1000; R&D Systems, Minneapolis, MN, USA), β-actin (1:3000; Beyotime Biotech, Beijing, China), H3 (1:30,000; Abcam, Cambridge, MA, USA) and H3K9me3 (1:3000; Abcam). Blots were washed three times with phosphate-buffered saline (PBS), and then incubated with a horseradish peroxidase-conjugated anti-mouse secondary antibody (1:3000; Gene Tex, San Diego, CA, USA) or anti-rabbit secondary antibody (1:3000; Abcam). Protein bands of interest were visualized using an Image Quant ECL system (GE Healthcare, Piscataway, NJ, USA).

Immunofluorescence labeling of γ-H2AX foci

Cells were passaged onto slides, exposed 24 h later to 4 Gy of γ-irradiation, and incubated at 37 °C for 4 h. Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, washed again with PBS, permeabilized for 10 min using 0.05% Triton X-100 and 0.5% NP-40, and then washed three times (5 min each) in PBS. The cells were blocked for 1 h with 2% bovine serum albumin (BSA), and then incubated for 1 h at room temperature with a mouse anti-γH2AX antibody (1:500; Millipore, Temecula, CA, USA). Cells were washed three times with PBS containing 0.05% Tween 20, and then incubated with a goat anti-mouse secondary antibody (1:800; Abcam) for 1 h in the dark at room temperature. Cells were counterstained with 0.2 mg/mL 4′,6-diamidino-2-phenylindole (DAPI, 1:2000; Sigma, Shanghai, China). Confocal images were acquired and analyzed using a TCS SP5 (Leica) microscope equipped with an HCX PL 63 × 1.4 CS oil-immersion objective lens.

DNA extraction

Three types of cells (lv-iPSCs, ci-iPSCs, ESCs) were digested with 0.25% trypsin and re-suspended in gelatin-coated dishes. After incubation at 37 °C for 15 min, supernatants were transferred to 15-mL centrifuge tubes, and cells were collected by centrifugation at 500g for 5 min at room temperature. DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).

Whole-genome re-sequencing

Whole-genome DNA libraries suitable for sequencing using an Illumina sequencing platform were generated from 1-µg genomic DNA. The DNA was sheared to approximately 300–500 bp using a Covaris S220 instrument (Life Technologies, Carlsbad, CA, USA). A total of 2× 101-bp paired-end reads were produced using the HiSeq 2000 DNA Sequencer.

The sequencing data were mapped to a reference mouse genomic sequence (mm9) using the Burrows–Wheeler alignment tool algorithm [31]. Unique alignment reads were retained for later analysis. Using the untreated cells as a control, single-nucleotide variations (SNVs) were collected using the “mpileup” tool in SAMTools as well as the UnifiedGenotyper in the GATK module [32, 33]. Quality recalibration and local realignment were performed using GATK tools before variation calling was performed. The following criteria were applied for calling mutations using pairwise samples: (1) the minimum coverage of variant sites had to be greater than 20 and base quality greater than 15; (2) the frequency of mutant SNVs had to be 0 in control samples and 0.2 in irradiated samples; and (3) the variant sites had to be supported by at least two reads on the forward strand and two reads on the reverse strand.

RNA sequencing

Total RNA was extracted from each cell line using TRIzol reagent and enriched for mRNA using oligo (dT) magnetic beads. Approximately 1-µg mRNA was fragmented and electrophoresed to isolate mRNA fragments (200–250 bases). These fragments were subjected to end repair, 3′ terminal adenylation and adapter ligation, followed by cDNA synthesis. The resulting cDNAs were gel-electrophoresed to isolate 250–300 bp fragments, and were sequenced using a HiSeq 2000 system (Illumina).

Sequencing reads were aligned to a reference sequence (GRCm37/mm9) using TopHat alignment software [34, 35]. Only uniquely aligned reads were used for transcript assembly, which was performed using Cufflinks software [36]. Read counts for each gene were calculated, and the expression levels of each gene were normalized using the “fragments per kilobase of exon model per million mapped” (FPKM) algorithm. Differentially expressed genes were filtered based on false discovery rate (FDR)-adjusted P < 0.05. The profile of differentially expressed genes was visualized and analyzed using the Bioconductor function “CummeRbund” in the R program [37]. Hierarchical clustering was performed using the “heatmap” package in R.

Generation of iPSC- and ESC-derived mice

Two cell-stage ICR embryos were electrofused to produce tetraploid embryos, and 10–15 iPSCs and ESCs were subsequently injected into the reconstructed tetraploid blastocysts. Embryos were cultured for 1 day prior to transplantation into the uterus of pseudo-pregnant mice. Caesarean sections were performed at E19.5, and the pups were fostered by lactating ICR mothers [38].

Comet assay

Mice derived from iPSCs or ESCs as well as C57 mice were treated with 4 Gy ionizing radiation. At 4 h later, bone marrow cells were isolated and re-suspended using PBS and concentrated by adding 150-μL molten 0.75% low-melting-point agarose. An aliquot of concentrated cells (60 μL) was then added to molten 0.8% normal-melting-point agarose on comet slides. The slides were incubated for 1–2 h at 4 °C with pre-chilled lysis buffer, stored in the dark at 4 °C for 20 min, then incubated with pre-chilled electrophoresis buffer (0.3 mol/L NaOH containing 0.5 mol/L EDTA, pH > 13.0). Gel electrophoresis was performed at 25 V for 20 min at 4 °C. Slides were incubated at 4 °C for 15 min with neutralization buffer (0.4 mol/L Tris, pH > 7.5), washed with 100% ethanol for 3–5 min and air-dried at room temperature. Diluted ethidium bromide (EB) solution (20–30 μL) was placed onto each dried agarose circle. Slides were then read at 100 cells/sample using a fluorescence microscope equipped with CASP DNA damage analysis software.

Results

Similar gene expression profile between lv-iPSCs and ESCs

RNA-seq analysis showed that the gene expression profile of lv-iPSCs was similar to that of ESCs but not to that of MEFs (Fig. 1a), indicating iPSC pluripotency. Since genomic stability depends on DNA damage repair, we analyzed expression of the genes involved in DNA damage repair pathways. No significant differences in the expression of such genes were found between lv-iPSCs and ESCs (Fig. 1b). We further analyzed the expression of DNA repair genes that were identified during early reprogramming of iPSCs in our previous report [39] and confirmed the up-regulation of those genes at early iPSC stages (Fig. 1c). These results suggest that DNA damage repair pathways can be reprogrammed at early iPSC stages and become similar to pathways in ESCs as reprogramming continues [39].

Fig. 1
figure 1

Gene expression profile of ESCs, lv-iPSCs and MEFs. a Scatter plots used to identify global trends in gene expression and differences among cell lines. b Heat maps showing the expression level of DNA damage repair-associated genes in the cell lines. Blue color indicates lowest expression; fuchsia, highest. c Re-analysis of the expression of DNA damage repair-associated genes during early reprogramming

Full size image

More DNA mutations in lv-iPSCs than in other cell types after ionizing irradiation

We treated mouse lv-iPSCs, ESCs and MEFs with 4 Gy ionizing radiation to induce double-strand breaks. If not repaired properly, such breaks can result in genomic abnormalities, apoptosis and senescence [23, 26, 40]. Whole-genome DNA sequencing at 4 h after irradiation revealed more SNVs in lv-iPSCs than in the other cell types (Fig. 2a, Table 1), as well as more short indels (Fig. 2a, Table 2). MEFs showed a larger variety of copy number variations (CNVs) than the other cell types (Fig. 2a).

Fig. 2
figure 2

Genomic variation in each cell line after ionizing irradiation. a Circos plot showing genetic alterations in lv-iPSCs, ESCs and MEFs after irradiation, based on the corresponding untreated cells as the reference. CHR, chromosome. b, c Histograms showing the numbers of (b) single-nucleotide variations (SNVs) and (c) short insertions or deletions (indels) in each type of genomic region in each cell line. d Histogram of the number of SNVs in a coding region (CDS) in each cell line

Full size image
Table 1 Summary of sequencing results
Full size table
Table 2 Summary of somatic indels in each cell line
Full size table

A larger number of SNVs and indels occurred in coding regions, intergenic regions, introns, 5′ untranslated regions (UTRs) and 3′ UTRs of lv-iPSCs than in other cell types (Fig. 2b, c). Irradiation was associated with the appearance of many more synonymous point mutations in coding regions in lv-iPSCs (559) than in ESCs (8) or MEFs (11) (Fig. 2d, Table 3). Similarly, many more non-synonymous point mutations in coding regions were found in lv-iPSCs (307) than in ESCs (7) or MEFs (13) (Fig. 2d, Tables 3, 4, 5, 6).

Table 3 Summary of somatic mutations in each cell line
Full size table
Table 4 Frequencies of coding SNVs in ESCs exposed to ionizing radiation
Full size table
Table 5 Frequencies of coding SNVs in MEFs exposed to ionizing radiation
Full size table
Table 6 Frequencies of coding SNVs in lv-iPSCs exposed to ionizing radiation
Full size table

Similar gene expression profile in lv-iPSCs with or without ionizing radiation

To determine whether ionizing radiation alters the expression of certain genes in lv-iPSCs that may help explain the high mutation rate, RNA-seq analysis was conducted in irradiated versus control cells. The results indicated a similar gene expression profile with or without radiation (Fig. 3a). In fact, irradiation appeared to up-regulate only 46 genes in ESCs and 30 genes in lv-iPSCs (Fig. 3b). In contrast to the genes in lv-iPSCs that radiation up-regulated, majority of the genes up-regulated in ESCs is implicated in cellular response to stress and cell cycle processes (Fig. 3c, d).

Fig. 3
figure 3

Gene expression levels in cells exposed or not to ionizing radiation (IR) for the indicated periods. a Heatmap showing Pearson’s correlation coefficients relating expression levels between irradiated and non-irradiated cells. b Volcano plots of genes expressed in irradiated and non-irradiated cells, showing genes significantly up-regulated (red dots) or down-regulated (green dots) in irradiated cells. Differentially expressed genes were filtered based on FDR < 0.05. c, d Histograms of gene ontology classifications of differentially expressed genes in irradiated (c) ESCs and (d) iPSCs. e Heat maps showing the expression level of DNA damage repair-associated genes in irradiated (+) and non-irradiated (−) cells. Blue indicates lowest expression; fuchsia, highest. BER base excision repair, HR, homologous recombination, MMR mismatch repair, NER nucleotide excision repair, NHEJ non-homologous end joining

Full size image

Expression levels of genes involved in DNA damage repair pathways were higher in lv-iPSCs and ESCs than in MEFs, and ionizing radiation did not substantially alter the expression of these genes (Fig. 3e). Thus the genomic instability of lv-iPSCs is unlikely to reflect changes in the expression level of genes involved in DNA damage repair.

Weaker DNA damage repair response to ionizing radiation in lv-iPSCs

The phosphorylated histone variant H2AX (γ-H2AX) is a marker of double-strand breaks. Ionizing radiation significantly increased the number of γ-H2AX foci in lv-iPSCs, ESCs and MEFs, but the magnitude of decrease was much smaller in lv-iPSCs (Fig. 4a), suggesting lower capacity to repair DNA damage.

Fig. 4
figure 4

The phosphorylation level of DNA repair-associated proteins. a Quantification of the numbers of γ-H2AX foci in lv-iPSCs, ESCs and MEFs. Error bars represent the standard error of the mean (SEM) for the numbers of γ-H2AX foci per nucleus based on 4–5 fields, each containing approximately 20–30 cells. Significance of differences was assessed using Student’s t test. **P < 0.01 (three independent experiments). b Western blot analysis of phosphorylated ATM (p-ATM) and phosphorylated catalytic subunit of DNA protein kinase (p-DNA-PKcs) in lv-iPSCs and ESCs before and after ionizing irradiation. c Western blot analysis of the trimethylation level of H3K9 in lv-iPSCs and ESCs before and after ionizing irradiation

Full size image

Next we tested whether the lower genomic stability of lv-iPSCs reflects deficiency in the error-free HR repair pathway. Indeed, we found ATM phosphorylation to be defective in lv-iPSCs (Fig. 4b) [30, 41]. We also found lower levels of H3K9me3, which recruits repair proteins to double-strand breaks, in irradiated lv-iPSCs than in irradiated ESCs or MEFs (Fig. 4c). All together, these findings may help explain the higher mutation rate of lv-iPSCs.

Lower genomic stability in lv-iPSCs than ci-iPSCs

Treatment with ionizing radiation led to higher levels of phosphorylated ATM in ci-iPSCs than in lv-iPSCs (Fig. 5a). This may help explain the higher genomic stability of ci-iPSCs [41]. Whole-genome re-sequencing at 4 h after irradiation revealed 1709 SNVs in the ci-iPSCs; this was slightly more than in treated ESCs but far less than in lv-iPSCs (Fig. 5b). Similarly, the proportion of SNVs in coding sequences, introns, 5′ or 3′ UTRs and intergenic regions was slightly higher in ci-iPSCs than in ESCs, but much higher in lv-iPSCs (Fig. 5c, d). These results indicate greater genomic stability in ci-iPSCs than in lv-iPSCs, which is due at least in part to greater activity of the HR pathway of DNA damage repair.

Fig. 5
figure 5

High genome stability of ci-iPSCs. a Western blot analysis of phosphorylated ATM (p-ATM) in ci-iPS, lv-iPS and ESCs before and after ionizing irradiation. b Circos plot showing genetic alterations in irradiated ci-iPSCs and ESCs, based on the corresponding untreated cells as a reference. Chromosome numbers are indicated as the outermost labels. c Histograms showing the number of SNVs in each genomic region of irradiated lv-iPSCs, ci-iPSCs and MEFs. CDS coding sequence, SNV single-nucleotide variants, UTR untranslated region. d Histograms showing the numbers of SNVs in the coding regions of irradiated lv-iPSCs, ci-iPSCs and MEFs

Full size image

lv-iPSCs can tolerate more genomic DNA variation

The abovementioned results led us to hypothesize that lv-iPSCs can survive with greater genomic variation than the other cell types. Consistent with this hypothesis, we found that lv-iPSCs indeed had more DNA variation than the other cell types, yet the percentage of apoptotic lv-iPSCs did not increase between 24 and 48 h after irradiation (Fig. 6a) and the rate of lv-iPSC proliferation was greater than that of ESCs or MEFs (Fig. 6b). When we analyzed whether irradiation arrested lv-iPSCs in the G2/M phase, we observed a high proportion of arrested cells at 24 h after irradiation, but a lower proportion at 48 h (Fig. 6c). We observed similar results with ESCs, showing an increased proportion of ESCs in G2/M phase at 24 h after irradiation and a lower radiation arrest at 48 h. These results suggest that lv-iPSCs tolerate greater genomic DNA variation than the other cell types.

Fig. 6
figure 6

Greater tolerance of genomic DNA variation in lv-iPSCs. a Flow cytometric analysis of apoptosis rate in lv-iPSCs, ESCs and MEFs following ionizing irradiation (IR) for the indicated periods. 7-AAD 7-amino-actinomycin. b Cell proliferation rate (based on BrdU incorporation) in lv-iPSCs, ESCs and MEFs exposed to ionizing irradiation for the indicated periods. Each point represents a mean of three replicates **P < 0.01. c Analysis of cell cycle distribution in lv-iPS, ESCs and MEFs exposed to ionizing radiation for the indicated periods

Full size image

lv-iPSCs are more susceptible to DNA damage

Next we compared genomic stability in mice derived from lv-iPSCs versus ESCs. C57 mice were included as additional control. Irradiation of the mice led to a higher percentage of impaired bone marrow cells (Fig. 7a–c) and of tail DNA in bone marrow cells (Fig. 7d) in iPSC-derived mice than in ESC-derived mice and C57 mice. These results suggest that mice derived from lv-iPSCs have lower DNA damage repair capability than ESC-derived or C57 mice and are therefore more susceptible to DNA damage.

Fig. 7
figure 7

Genome stability of mice derived from lv-iPSCs or ESCs following exposure to ionizing radiation (IR). Controls were C57 mice. a Mice were generated from lv-iPSCs or ESCs through tetraploid embryo complementation. Representative results from three independent experiments are shown. b Examples of bones from the three types of mice, from which marrow cells were extracted. c Box plots showing the percentage of impaired bone marrow cells in each mouse strain. DNA damage was evaluated using single-cell gel electrophoresis **P < 0.01. d Box plots showing the percentage of Tail DNA in impaired cells as a measure of DNA damage. Tail DNA% = Tail DNA intensity/Cell DNA Intensity × 100%. CASP software was used to calculate tail moment based on 50–100 randomly selected cells per sample

Full size image

Taken together, our in vitro and in vivo experiments suggest that lv-iPSCs are more sensitive to environmental stress than ci-iPSCs, ESCs or MEFs. Ionizing radiation induces higher genomic mutation rates in lv-iPSCs, which nevertheless better tolerate the resulting genomic alterations. Genomic mutations that accumulate in lv-iPSCs are passed onto the next generation, resulting in genomic instability (Fig. 8).

Fig. 8
figure 8

Diagram illustrating factors influencing the genome stability of iPSCs. Environmental factors contribute to genomic variations in lv-iPSCs. In response to double-strand DNA breaks, lv-iPSCs always adopt the error-prone NHEJ repair pathway. The resulting low fidelity of DNA repair makes the lv-iPSC genome unstable and the cells more vulnerable to environmental stress. Genomic stability of iPSCs appears to depend on the method used to generate them: ci-iPSCs show greater stability than lv-iPSCs. HR homologous recombination repair pathway, IR ionizing radiation, NHEJ non-homologous end joining

Full size image

Discussion

Reprogramming to generate iPSCs more efficiently [29, 42,43,44,45,46,47,48,49,50,51] has been linked to the accumulation of genomic abnormalities [52,53,54,55,56,57,58,59]. This poses a problem for the use of iPSCs, since mice derived from such cells can tolerate the accumulation of somatic mutations for up to six generations [60]. In the present study, we used whole-genome sequencing to compare the genomic stability of iPSCs prepared using lentivirus or chemically, and to benchmark that stability against ESCs and MEFs. We found that ionizing irradiation led to the highest rate of somatic mutations and short indels in lv-iPSCs, and this correlated with low levels of ATM phosphorylation, indicating low fidelity of DNA damage repair [41]. Experiments in vitro and in mice derived from lv-iPSCs showed that this type of pluripotent cell tolerates genomic mutations better than the other cell types evaluated.

Although iPSCs resemble ESCs in morphology, gene expression profile and in vitro differentiation capacity, they differ substantially in genomic stability. The low fidelity of DNA repair observed in our study suggests that irradiation of lv-iPSCs induces a high rate of genomic abnormalities, which is less likely to trigger apoptosis in these cells and is therefore more likely to be tolerated, thus leading to a high rate of tumorigenesis in vivo. Compromised error-free HR pathway of DNA damage repair in lv-iPSCs may help explain the relatively high genomic instability in these cells. Indeed, inhibiting the HR pathway in iPSCs has been shown to destabilize the genome [61].

Our results suggest that the epigenetic status of iPSCs may contribute to, or modulate, their genomic instability. Variation in levels of H3K9me3 and phosphorylated ATM among iPSCs may mean that cells vary in their reliance on DNA damage repair pathways, which vary in their fidelity. Future studies should further examine the potential involvement of epigenetics and other factors in iPSC genomic instability.

Future work is also needed to clarify to what extent factors that are intrinsic or extrinsic to stem cells determine the risk of malignant transformation. Tomasetti et al. found that cancer risk in certain tissues correlated strongly with the number of divisions that the stem cells had undergone, suggesting that the accumulation of genomic mutations is primarily responsible for high risk of tumorigenesis [62]. Another study, in contrast, suggested that intrinsic factors account for only 10%–30% of cancer risk, with the majority of the risk due to extrinsic factors [63]. The results from the present study suggest that extrinsic factors induce more genomic mutations than intrinsic factors in lv-iPSCs. The high rate of tumorigenesis of iPSCs in vivo suggests that extrinsic factors strongly contribute to cancer risk and carcinogenesis.

Conclusions

The present study demonstrated a low level of DNA damage repair in iPSCs. Ionizing radiation induced more somatic mutations and short indels in iPSCs than in ESCs or MEFs. Genome stability was higher in iPSCs induced chemically than in iPSCs induced with lentivirus. The high genome instability of lv-iPSCs appears to reflect increased NHEJ and decreased HR pathways of DNA damage repair, and could contribute to the high rate of tumorigenesis in vivo.

References

  1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282(5391):1145–7.

    Article  PubMed  CAS  Google Scholar 

  2. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–76.

    Article  PubMed  CAS  Google Scholar 

  3. de Lazaro I, Yilmazer A, Kostarelos K. Induced pluripotent stem (iPS) cells: a new source for cell-based therapeutics? J Control Release. 2014;185:37–44.

    Article  PubMed  Google Scholar 

  4. Amabile G, Meissner A. Induced pluripotent stem cells: current progress and potential for regenerative medicine. Trends Mol Med. 2009;15(2):59–68.

    Article  PubMed  CAS  Google Scholar 

  5. Yamanaka S. A fresh look at iPS cells. Cell. 2009;137(1):13–7.

    Article  PubMed  CAS  Google Scholar 

  6. Meyer N, Penn LZ. Reflecting on 25 years with MYC. Nat Rev Cancer. 2008;8(12):976–90.

    Article  PubMed  CAS  Google Scholar 

  7. Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet. 2008;40(5):499–507.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Malchenko S, Galat V, Seftor EA, Vanin EF, Costa FF, Seftor RE, et al. Cancer hallmarks in induced pluripotent cells: new insights. J Cell Physiol. 2010;225(2):390–3.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Rowland BD, Peeper DS. KLF4, p21 and context-dependent opposing forces in cancer. Nat Rev Cancer. 2006;6(1):11–23.

    Article  PubMed  CAS  Google Scholar 

  10. Ghosh Z, Huang M, Hu S, Wilson KD, Dey D, Wu JC. Dissecting the oncogenic and tumorigenic potential of differentiated human induced pluripotent stem cells and human embryonic stem cells. Cancer Res. 2011;71(14):5030–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Blasco MA, Serrano M, Fernandez-Capetillo O. Genomic instability in iPS: time for a break. EMBO J. 2011;30(6):991–3.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Pasi CE, Dereli-Oz A, Negrini S, Friedli M, Fragola G, Lombardo A, et al. Genomic instability in induced stem cells. Cell Death Differ. 2011;18(5):745–53.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Pera MF. Stem cells: the dark side of induced pluripotency. Nature. 2011;471(7336):46–7.

    Article  PubMed  CAS  Google Scholar 

  14. Ronen D, Benvenisty N. Genomic stability in reprogramming. Curr Opin Genet Dev. 2012;22(5):444–9.

    Article  PubMed  CAS  Google Scholar 

  15. Sarig R, Rotter V. Can an iPS cell secure its genomic fidelity? Cell Death Differ. 2011;18(5):743–4.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. von Joest M, Bua Aguin S, Li H. Genomic stability during cellular reprogramming: mission impossible? Mutat Res. 2016;788:12–6.

    Article  Google Scholar 

  17. Friedberg EC. A history of the DNA repair and mutagenesis field The discovery of base excision repair. DNA Repair. 2016;37:A35–9.

    Article  PubMed  CAS  Google Scholar 

  18. Lieber MR. The mechanism of human nonhomologous DNA end joining. J Biol Chem. 2008;283(1):1–5.

    Article  PubMed  CAS  Google Scholar 

  19. Filippo JS, Sung P, Klein H. Mechanism of eukaryotic homologous recombination. Annu Rev Biochem. 2008;77:229–57.

    Article  Google Scholar 

  20. Weeden CE, Chen YS, Ma SB, Hu YF, Ramm G, Sutherland KD, et al. Lung basal stem cells rapidly repair DNA damage using the error-prone nonhomologous end-joining pathway. PLoS Biol. 2017;15(1):e2000731.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Long Y, Wang M, Gu HF, Xie X. Bromodeoxyuridine promotes full-chemical induction of mouse pluripotent stem cells. Cell Res. 2015;25(10):1171–4.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Jackson SP. Sensing and repairing DNA double-strand breaks. Carcinogenesis. 2002;23(5):687–96.

    Article  PubMed  CAS  Google Scholar 

  23. Khanna KK, Jackson SP. DNA double-strand breaks: signaling, repair and the cancer connection. Nat Genet. 2001;27(3):247–54.

    Article  PubMed  CAS  Google Scholar 

  24. Lombard DB, Chua KF, Mostoslavsky R, Franco S, Gostissa M, Alt FW. DNA repair, genome stability, and aging. Cell. 2005;120(4):497–512.

    Article  PubMed  CAS  Google Scholar 

  25. Zha S, Alt FW, Cheng HL, Brush JW, Li G. Defective DNA repair and increased genomic instability in Cernunnos-XLF-deficient murine ES cells. Proc Natl Acad Sci USA. 2007;104(11):4518–23.

    Article  PubMed  CAS  Google Scholar 

  26. Rooney S, Alt FW, Lombard D, Whitlow S, Eckersdorff M, Fleming J, et al. Defective DNA repair and increased genomic instability in artemis-deficient murine cells. J Exp Med. 2003;197(5):553–65.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Esteban MA, Wang T, Qin BM, Yang JY, Qin DJ, Cai JL, et al. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell. 2010;6(1):71–9.

    Article  PubMed  CAS  Google Scholar 

  28. Zhang M, Yang C, Liu H, Sun Y. Induced pluripotent stem cells are sensitive to DNA damage. Genomics Proteomics Bioinf. 2013;11(5):320–6.

    Article  CAS  Google Scholar 

  29. Huangfu DW, Maehr R, Guo WJ, Eijkelenboom A, Snitow M, Chen AE, et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol. 2008;26(7):795–7.

    Article  PubMed  CAS  Google Scholar 

  30. Sun Y, Xu Y, Roy K, Price BD. DNA damage-induced acetylation of lysine 3016 of ATM activates ATM kinase activity. Mol Cell Biol. 2007;27(24):8502–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25(14):1754–60.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25(16):2078–9.

    Article  PubMed  PubMed Central  Google Scholar 

  33. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20(9):1297–303.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013;14(4):R36.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7(3):562–78.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004;5(10):R80.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Zhao XY, Lv Z, Li W, Zeng F, Zhou Q. Production of mice using iPS cells and tetraploid complementation. Nat Protoc. 2010;5(5):963–71.

    Article  PubMed  CAS  Google Scholar 

  39. Shu J, Zhang K, Zhang MJ, Yao AZ, Shao SD, Du FX, et al. GATA family members as inducers for cellular reprogramming to pluripotency. Cell Res. 2015;25(2):169–80.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Johnson RD, Jasin M. Double-strand-break-induced homologous recombination in mammalian cells. Biochem Soc Trans. 2001;29(Pt 2):196–201.

    Article  PubMed  CAS  Google Scholar 

  41. Lavin MF, Birrell G, Chen P, Kozlov S, Scott S, Gueven N. ATM signaling and genomic stability in response to DNA damage. Mutat Res. 2005;569(1–2):123–32.

    Article  PubMed  CAS  Google Scholar 

  42. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861–72.

    Article  PubMed  CAS  Google Scholar 

  43. Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K. Induced pluripotent stem cells generated without viral integration. Science. 2008;322(5903):945–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature. 2009;458(7239):771–5.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hamalainen R, et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature. 2009;458(7239):766–70.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Zhou W, Freed CR. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells. 2009;27(11):2667–74.

    Article  PubMed  CAS  Google Scholar 

  47. Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, et al. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell. 2009;4(5):381–4.

    Article  PubMed  CAS  Google Scholar 

  48. Warren L, Manos PD, Ahfeldt T, Loh Y-H, Li H, Lau F, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 2010;7(5):618–30.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Miyoshi N, Ishii H, Nagano H, Haraguchi N, Dewi DL, Kano Y, et al. Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell. 2011;8(6):633–8.

    Article  PubMed  CAS  Google Scholar 

  50. Hou PP, Li YQ, Zhang X, Liu C, Guan JY, Li HG, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. 2013;341(6146):651–4.

    Article  PubMed  CAS  Google Scholar 

  51. Shu J, Wu C, Wu Y, Li Z, Shao S, Zhao W, et al. Induction of pluripotency in mouse somatic cells with lineage specifiers. Cell. 2013;153(5):963–75.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Stadtfeld M, Apostolou E, Akutsu H, Fukuda A, Follett P, Natesan S, et al. Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature. 2010;465(7295):175–81.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Laurent LC, Ulitsky I, Slavin I, Tran H, Schork A, Morey R, et al. Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell. 2011;8(1):106–18.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Mayshar Y, Ben-David U, Lavon N, Biancotti JC, Yakir B, Clark AT, et al. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell. 2010;7(4):521–31.

    Article  PubMed  CAS  Google Scholar 

  55. Lister R, Pelizzola M, Kida YS, Hawkins RD, Nery JR, Hon G, et al. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature. 2011;471(7336):68–73.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Polo JM, Liu S, Figueroa ME, Kulalert W, Eminli S, Tan KY, et al. Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nat Biotechnol. 2010;28(8):848–55.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, et al. Somatic coding mutations in human induced pluripotent stem cells. Nature. 2011;471(7336):63–7.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, et al. Epigenetic memory in induced pluripotent stem cells. Nature. 2010;467(7313):285–90.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Kim K, Zhao R, Doi A, Ng K, Unternaehrer J, Cahan P, et al. Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells. Nat Biotechnol. 2012;30(1):1117–9.

    Article  Google Scholar 

  60. Gao S, Zheng C, Chang G, Liu W, Kou X, Tan K, et al. Unique features of mutations revealed by sequentially reprogrammed induced pluripotent stem cells. Nat Commun. 2015;6:6318.

    Article  PubMed  CAS  Google Scholar 

  61. Thompson LH, Schild D. The contribution of homologous recombination in preserving genome integrity in mammalian cells. Biochimie. 1999;81(1–2):87–105.

    Article  PubMed  CAS  Google Scholar 

  62. Tomasetti C, Vogelstein B. Cancer etiology. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science. 2015;347(6217):78–81.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Wu S, Powers S, Zhu W, Hannun YA. Substantial contribution of extrinsic risk factors to cancer development. Nature. 2016;529(7584):43–7.

    Article  PubMed  CAS  Google Scholar 

Download references

Authors’ contributions

YS, QZ and JC conceived this study. MZ and LW contributed to its design. MZ, GL, KA, CY, HL, FD, XH and YL performed experiments, while MZ and GL performed bioinformatic analyses. JC assisted with bioinformatic analysis and interpretation. YS and MZ interpreted the data and wrote the paper with the assistance of the other authors. QZ, DP, and XX provided critical technical assistance and expertise. All authors read and approved the final manuscript.

Acknowledgements

We thank Professor Qi Zhou from the Institute of Zoology of the Chinese Academy of Sciences for generously supplying iPS- and ES-derived mice. We thank Professor Duanqing Pei from the Guangzhou Institutes of Biomedicine and Health of the Chinese Academy of Sciences for providing the lv-iPSC line, and Xin Xie from the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences for providing the ci-iPSC line. We are also grateful to our laboratory colleagues for their assistance with experiments and manuscript preparation.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The raw sequencing data reported in this manuscript are publicly available at the Genome Sequence Archive (http://gsa.big.ac.cn) under Accession Number CRA000695.

All data generated and analyzed are included in the article to support the conclusions.

Consent for publication

Not applicable.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Beijing Institute of Genomics and the School of Life Sciences at the Chinese Academy of Sciences.

Funding

This work was supported by the Precision Medicine Research Program of the Chinese Academy of Sciences (KJZD-EW-L14), Strategic Priority Research Program of the Chinese Academy of Sciences (XDA01040407), National Natural Science Foundation of China (31471395, 91019024, 31540033 and 31100558), National Basic Research Program of China (973 Program, 2012CB518302 and 2013CB911001) and 100 Talents Project.

Author information

Author notes

  1. Minjie Zhang, Liu Wang and Ke An are co-first authors and contributed equally to this work

Authors and Affiliations

  1. Key Laboratory of Genomic and Precision Medicine, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, P. R. China

    Minjie Zhang, Ke An, Jun Cai, Guochao Li, Caiyun Yang, Huixian Liu, Fengxia Du, Xiao Han, Zilong Zhang, Zitong Zhao & Yingli Sun

  2. University of Chinese Academy of Sciences, Beijing, 100049, P. R. China

    Minjie Zhang, Ke An, Guochao Li, Xiao Han, Zilong Zhang & Zitong Zhao

  3. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, P. R. China

    Liu Wang & Qi Zhou

  4. The Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, P. R. China

    Duanqing Pei

  5. CAS Key Laboratory of Receptor Research, the National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, P. R. China

    Yuan Long & Xin Xie

Authors
  1. Minjie Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Liu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ke An
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jun Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Guochao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Caiyun Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Huixian Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Fengxia Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Xiao Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Zilong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Zitong Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Duanqing Pei
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Yuan Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Xin Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Qi Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Yingli Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yingli Sun.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, M., Wang, L., An, K. et al. Lower genomic stability of induced pluripotent stem cells reflects increased non-homologous end joining. Cancer Commun 38, 49 (2018). https://doi.org/10.1186/s40880-018-0313-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s40880-018-0313-0

Keywords

  • Genomic stability
  • DNA damage repair
  • iPSCs
  • ESCs

Cancer Communications

ISSN: 2523-3548

Contact us